DETECTION AND CHARACTERIZATION OF RICKETTSIAE IN WESTERN AUSTRALIA. Helen Clare OWEN, BVMS

Transcription

1 DETECTION AND CHARACTERIZATION OF RICKETTSIAE IN WESTERN AUSTRALIA Helen Clare OWEN, BVMS This thesis is presented for the degree of Doctor of Philosophy of Murdoch University, 2007.

2 I declare that this thesis is my own account of my research and contains as its main content work which has not previously been submitted for a degree at any tertiary education institution Helen Clare OWEN ii

3 ABSTRACT The aim of this study was to address the shortfall in current, in-depth knowledge of Western Australian rickettsiae. Historically, murine typhus had been extensively reported and, more recently, serological studies and a small number of diagnosed cases indicated that spotted fever group rickettsiae were also present in the State, however no attempts had been made to isolate or characterize these rickettsiae. To facilitate investigation, ectoparasites (principally ticks) were opportunistically collected from across the State, with an emphasis on native and feral animals and people. All ectoparasites were screened for rickettsial infection using a polymerase chain reaction incorporating Rickettsia-specific citrate synthase gene (glta) primers. Preliminary sequencing was performed on representative PCR-positive samples from each geographical location, vertebrate host and ectoparasite in order to identify and characterize the infecting rickettsia. Isolation in cell culture and further genotypic characterization was then performed. Finally, a serosurvey and questionnaire were implemented in one of the study areas to determine whether people were being infected with a Rickettsia spp. and whether infection was associated with clinical signs. Ectoparasite collection produced three genera of ticks (Ixodes, Amblyomma and Haemaphysalis) from native animals, feral pigs and people, primarily from the southwest of Western Australia and Barrow Island in the Pilbara region. Ticks from a number of sources were shown to be infected with rickettsiae by the PCR, including feral pigs, people, bobtail lizards, kangaroos, bandicoots, burrowing bettongs, iii

4 common brushtail possums and yellow-footed antechinus. Genotypic characterization of positive amplicons from ticks revealed the presence of two novel spotted fever group rickettsiae. Rickettsia gravesii sp. nov., named in honour of Dr Stephen Graves, was identified extensively throughout the southwest of the State and on Barrow Island in Ixodes, Amblyomma and Haemaphysalis spp. ticks from multiple hosts. Candidatus Rickettsia antechini was detected in Ixodes spp. only from yellow-footed antechinus in Dwellingup. In addition, a novel Bartonella spp. (Bartonella sp. strain Mu1) was also detected from Acanthopsylla jordani fleas collected from yellow-footed antechinus in Dwellingup. Rickettsia gravesii sp. nov. is most closely related to the Rickettsia massiliae subgroup of the spotted fever group and to R. rhipicephali in particular. Sequence similarities between this novel species and the subgroup were 99.7%, 98.4%, 95.8% and 97.4% based on its 16S rrna, glta, ompa and ompb genes respectively. Candidatus Rickettsia antechini also demonstrated a close relationship to the R. massiliae subgroup (99.4%, 94.8% and 97.1% sequence similarity based on its glta, ompa and ompb genes respectively). The two novel Western Australian species demonstrated 98.4%, 96.3% and 96.7% sequence similarity to each other based on glta, ompa and ompb genes respectively indicating separate species. The novel Bartonella spp. (Bartonella sp. strain Mu1) detected in fleas collected from yellowfooted antechinus in Dwellingup demonstrated greatest glta gene sequence similarity to Bartonella strain 40 at 86.1%. Results from the serosurvey and questionnaire-based investigation into the zoonotic importance of R. gravesii sp. nov. on Barrow Island supported the results of the tick iv

5 study and suggested that a tick-borne rickettsia(e) was infecting people on the island. However, a significant association between seroconversion and a history of symptoms consistent with a rickettsiosis was not found, and it is possible therefore, that R. gravesii sp. nov. produces only asymptomatic infections. Future work on rickettsiae in Western Australia will involve phenotypic characterization of the novel species, further investigation of their epidemiology and pathogenicity and an ongoing search for additional undiscovered species. v

6 TABLE OF CONTENTS Title page i Declaration..ii Abstract...iii Table of Contents vi List of Tables.xii List of Figures xiii Acknowledgements xvi CHAPTER 1. GENERAL INTRODUCTION 1.1 ORDER RICKETTSIALES Introduction Taxonomy Epidemiology The association between ectoparasites and rickettsiae Vertebrate hosts and the effects of rickettsial infection Public health significance of rickettsial infections DIAGNOSIS OF RICKETTSIAL INFECTION Serology Culture of rickettsiae PCR detection of rickettsial DNA CHARACTERIZATION OF NOVEL RICKETTSIAE Antigenic and serological characterization Molecular-biological assays for identification. 16 vi

7 1.4 REVIEW OF RICKETTSIOSES Typhus group Epidemic typhus Murine/endemic typhus Spotted fever group Rocky Mountain spotted fever Mediterranean spotted fever Queensland tick typhus Flinders Island spotted fever Rickettsia felis Australian spotted fever Unidentified spotted fever group rickettsiae in Australia Other pathogenic spotted fever group rickettsiae Rickettsiae of unknown pathogenicity Non-pathogenic spotted fever group rickettsiae Scrub typhus Bartonella spp OBJECTIVES 42 CHAPTER 2. COLLECTION AND IDENTIFICATION OF ECTOPARASITES 2.1 INTRODUCTION MATERIALS AND METHODS Sample collection Tick identification RESULTS DISCUSSION...54 vii

8 CHAPTER 3. DETECTION OF RICKETTSIAE 3.1 INTRODUCTION MATERIALS AND METHODS Study design Ectoparasite identification Detection of rickettsial DNA using PCR Extraction of DNA Screening PCR PCR product detection methods Sequencing Sequencing of glta and ompa genes Sequence analysis RESULTS Screening of ectoparasites Prevalence Sequence similarities DISCUSSION Aetiology Prevalence Life cycle: vectors and vertebrate hosts Geographical distribution Zoonotic potential..80 CHAPTER 4. CHARACTERIZATION OF RICKETTSIA spp. STRAIN BWI INTRODUCTION MATERIALS AND METHODS.84 viii

9 4.2.1 Sample collection Cell culture Immunofluorescence assay Sequencing Sequence analysis RESULTS Sequence analysis DISCUSSION.97 CHAPTER 5. DETECTION AND CHARACTERIZATION OF NOVEL RICKETTSIAE FROM YELLOW-FOOTED ANTECHINUS. 5.1 INTRODUCTION MATERIALS AND METHODS Sample collection Ectoparasite identification Cell culture DNA extraction PCR screening Sequencing Sequence analysis RESULTS Prevalence Sequence similarities DISCUSSION Aetiology Ecology..117 ix

10 CHAPTER 6. INVESTIGATING THE PATHOGENIC POTENTIAL OF RICKETTSIA GRAVESII AND THE RISK FACTORS FOR ZOONOTIC INFECTION. 6.1 INTRODUCTION MATERIALS AND METHODS Study design Questionnaire design Questionnaire administration Blood collection Analysis RESULTS Descriptive epidemiology Analysis of questionnaire results DISCUSSION Review of major findings Limitations of the study Public health implications of the study Recommendations for further research..134 CHAPTER 7. GENERAL DISCUSSION 7.1 INTRODUCTION FINDINGS OF THE STUDY IN RELATION TO AIMS FUTURE DIRECTIONS REFERENCES APPENDICES 190 APPENDIX 1. ABREVIATIONS x

11 APPENDIX 2. SAMPLE COLLECTION INSTRUCTIONS..192 APPENDIX 3. COMMON AND SCIENTIFIC NAMES 193 APPENDIX 4. GENBANK ACCESSION NUMBERS APPENDIX 5. GENBANK ACCESSION NUMBERS AND SEQUENCES OF THE NOVEL STRAINS APPENDIX 6. MINIMUM EVOLUTION AND MAXIMUM PARSIMONY TREES 199 APPENDIX 7. QUESTIONNAIRE. 217 APPENDIX 8. RESULTS OF THE SEROSURVEY xi

12 LIST OF TABLES Table 2.1. Hosts from which tick species were collected in Western Australia, the collection sites and the numbers of animals on which ticks were found Table 2.2 Tick species reported in Western Australia...55 Table 3.1. Tick samples collected from different hosts and locations in Western Australia and their screening PCR status Table 3.2. Percentage glta sequence similarity between the two strains identified in this study and those of previously characterised Rickettsia spp. 70 Table 3.3. Percentage ompa sequence similarity between the two strains identified in this study and those of previously characterised Rickettsia spp. 71 Table Primers used for PCR and sequencing of DNA from Ricketssia spp. strain BWI Table 4.2 Conditions and the premix used for the PCR reactions amplifying Rickettsia spp. BWI-1 DNA,,,,,,.. 88 Table 4.3. PCR cycling conditions used in all reactions.88 Table 4.5 The percentage similarity between Rickettsia sp. strain BWI-1 and the previously characterized rickettsial species 91 Table 5.1 Results of PCR screening of ticks and fleas collected from yellow-footed antechinuses in Dwellingup, WA Table 5.2. Comparison of Rickettsia sp. strain D-1 and previously characterized species of Rickettsia based on 3 genes Table 5.3. Comparison of Bartonella sp. strain Mu1 and previously characterized species of Bartonella based on glta gene sequences xii

13 Table 6.1 Description of the population of Barrow Island workers that responded to the questionnaire. 126 Table 6.2 Summary of odds ratios for exposure variables versus rickettsial serology 128 Table A.1 Rickettsial Genbank accession numbers..194 Table A.2 Bartonella sp. GenBank accession numbers Table A.3 Titration results for the serology performed on respondents to the Barrow Island questionnaire 235 LIST OF FIGURES Figure 1.1 Composite diagram of the life cycle of Rocky Mountain spotted fever, Rickettsialpox, and murine typhus...5 Figure 2.1. Native animal trapping on Barrow Island.47 Figure 2.2. Tick on the ear of a burrowing bettong. 47 Figure 2.3 Map of Western Australia showing sample collection sites...48 Figure 2.4 Map of southwest WA showing sample collection sites...49 Figure 2.5. Dorsal view of Amblyomma triguttatum collected from a person on Barrow Island...52 Figure 2.6. Ventral view of Haemophysalis ratti collected from a golden bandicoot on Barrow Island..53 Figure 2.7. Dorsal view of Amblyomma limbatum collected from a common brushtail possum on Barrow Island 53 xiii

14 Figure 2.8. Dorsal view of Ixodes antechini collected from a yellow-footed antechinus in Dwellingup..54 Figure 2.9 A map of Western Australia demonstrating previously recorded distribution of ticks collected in this study Figure 3.1 Distribution of the rickettsial isolates across WA Figure 4.1. Unrooted neighbour-joining tree based on 16S rrna sequences demonstrating the relationship between Rickettsia sp. BWI-1 and previously characterised rickettsiae Figure 4.2. Neighbour-joining tree based on glta gene sequences demonstrating the relationship between Rickettsia sp. BWI-1 and previously characterised rickettsiae..93 Figure 4.3. Neighbour-joining tree based on ompa gene sequences demonstrating the relationship between Rickettsia sp. BWI-1 and previously characterised rickettsiae..94 Figure 4.4. Neighbour-joining tree based on ompb gene sequences demonstrating the relationship between Rickettsia sp. BWI-1 and previously characterised rickettsiae..95 Figure 4.5. Neighbour-joining tree based on sca4 sequences demonstrating the relationship between Rickettsia sp. BWI-1 and previously characterised rickettsiae..96 Figure 5.1. Acanthopsylla jordani, lateral view 107 Figure 5.2. Neighbour-joining tree based on glta sequences demonstrating the relationship between Rickettsia sp. D-1 and previously characterised rickettsiae.111 Figure 5.3. Neighbour-joining tree based on ompa sequences demonstrating the relationship between Rickettsia sp. D-1 and previously characterised rickettsiae 112 Figure 5.4. Neighbour-joining tree based on ompb sequences demonstrating the relationship between Rickettsia sp. D-1 and previously characterised rickettsiae.113 Figure 5.5. Neighbour-joining tree of Bartonella spp. based on glta sequences demonstrating the relationship between Bartonella sp. strain Mu1 and previously characterised Bartonella spp xiv

15 Figure A.1. Minimum-evolution tree based on 16S rrna sequences (R. gravesii) 199 Figure A.2. Maximum-parsimony tree based on 16S rrna sequences (R. gravesii) Figure A.3. Minimum-evolution tree based on glta sequences (R. gravesii) Figure A.4. Maximum-parsimony tree based on glta sequences (R. gravesii) 202 Figure A.5. Minimum-evolution tree based on ompa sequences (R. gravesii) 203 Figure A.6. Maximum-parsimony tree based on ompa sequences (R. gravesii)..204 Figure A.7. Minimum-evolution tree based on ompb sequences (R. gravesii).205 Figure A.8. Maximum-parsimony tree based on ompb sequences (R. gravesii) Figure A.9. Minimum-evolution tree based on sca4 sequences (R. gravesii) Figure A.10 Maximum-parsimony tree based on sca4 sequences (R. gravesii) Figure A.11. Minimum-evolution tree based on glta sequences (R. antechini) Figure A.12. Maximum-parsimony tree based on glta sequences (R. antechini).210 Figure A.13. Minimum-evolution tree based on ompa sequences (R. antechini).211 Figure A.14 Maximum-parsimony tree based on ompa sequences (R. antechini) 212 Figure A.15 Minimum-evolution tree based on ompb sequences (R. antechini) Figure A.16. Maximum-parsimony tree based on ompb sequences (R. antechini) Figure A.17 Minimum-evolution tree based on glta sequences (Bartonella sp. strain Mu1) Figure A.18 Maximum-parsimony tree based on glta sequences (Bartonella sp. strain Mu1). 216 xv

16 I had a lot of help. Thank you to my principal supervisor, Associate Professor Stan Fenwick for obtaining funding, recruiting collaborators and for his infectious enthusiasm for the project. Thanks also to my Murdoch co-supervisors, Associate Professor Ian Robertson and Associate Professor Phillip Clark. Ian agreed to help despite being a self-confessed molecular-biology sceptic, his presence on my supervisory panel was a constant source of reassurance and thanks to Phil especially for his perceptive and often amusing editing of my thesis. Thanks also to Dr Angus Cook, School of Population Health, UWA who guided me closely through the analysis of the questionnaire and Yazid Abdad, my fellow PhD candidate who kindly allowed me to use his serology results in my analysis. I am also eternally thankful to everyone at The Australian Rickettsial Reference Laboratory. Thanks to my co-supervisor Dr John Stenos, colleague Dr Nathan Unsworth and Chelsea Nyugen for their extensive knowledge of all things rickettsial. John did a fantastic job of interstate supervising and Nathan performed much of the initial culturing of the Western Australia rickettsiae while trying to pass on some of his formidable skills to me. Thank you also to Dr Stephen Graves, the laboratory s director, who is such a knowledgeable and willing collaborator and who provided such a catchy name for our novel rickettsia. Thank you to Gorgon, in particular Russell Langdon for their interest in the project and for contributing a great deal towards its funding. Thank you to my wonderful friend Patchara Phuektes who introduced me to the foreign world of the laboratory with unlimited patience. Thank you to Russ Hobbs for helping me identify my ectoparasite samples and also to Dr Andrew Mikosa, Dr Peter Spencer and Dr Ryan Jeffries who kindly helped me with the phylogenetic component xvi

17 of my project. Dr Peter Adams also provided a lot of helpful advice. Thank you to Dr Liam O Connor, Dr Pierre-Edouard Fournier and Professor Didier Raoult for allowing me to spend time in their laboratories. Scientists from the Department of Environmental Conservation (formerly CALM) especially Keith Morris and Tony Start, Kanyana Wildlife Rehabilitation Centre, Damien Cancilla, Roberta Bencini, Harriet Mills and John Drummond were kind enough to provide me with samples and thank you to Rod Armistead, Maggie Lilith and Felicity Donaldson for letting me come on some very entertaining fieldtrips as well. I m fairly certain that I couldn t have gotten through this without all the moral support and welcome distractions provided by my lovely friends Peter Wai in and Maggie Lilith. Thank you also for the companionship provided by my office mates: Kim, Danka, Celia, Jordan and Nico, who usually let me win the war over the air conditioner controls. Thank you to my extra-curricular friends Karen, Emma, Leigh, Emma and Russell, Megs and everyone in the UQ veterinary pathology department for not having the first idea about what rickettsiae are and thank you to Judy and Jan for looking after me while I was working in Victoria. The largest chunk of my gratitude must go to my family for their unfaltering support, especially my Mum who can always lessen the high drama that seems to accompany all my life s endeavours. xvii